Presynaptic 5-HT(1B) receptor-mediated serotonergic inhibition of glutamate transmission in the bed nucleus of the stria terminalis

Abstract

Activation of neurons in the bed nucleus of the stria terminalis (BNST) plays a critical role in stress and anxiety-related behaviors. Previously, we have shown that serotonin (5-HT) can directly modulate BNST neuronal excitability by an action at postsynaptic receptors. In this study we built upon that work to examine the effects of 5-HT on excitatory neurotransmission in an in vitro rat BNST slice preparation. Bath application of 5-HT reversibly reduced the amplitude of evoked excitatory postsynaptic currents (eEPSCs). These effects were mimicked by the 5-HT(1B/D) receptor agonist, sumatriptan, and by the 5-HT(1B) receptor selective agonist, CP93129. Conversely, the effects of 5-HT and sumatriptan could be blocked by the 5-HT(1B) receptor-selective antagonist, GR55562. In contrast, the 5-HT(1A) receptor agonist 8-OH DPAT or antagonist WAY 100635 could not mimic or block the effect of 5-HT on eEPSCs. Together, these data suggest that the 5-HT-induced attenuation of eEPSCs was mediated by 5-HT(1B) receptor activation. Moreover, sumatriptan had no effect on the amplitude of the postsynaptic current elicited by pressure applied AMPA, suggesting a possible presynaptic locus for the 5-HT(1B) receptor. Furthermore, 5-HT, sumatriptan and CP93129 all increased the paired pulse ratio of eEPSCs while they concomitantly decreased the amplitude of eEPSCs, suggesting that these agonists act to reduce glutamate release probability at presynaptic locus. Consistent with this observation, sumatriptan decreased the frequency of miniature EPSCs, but had no effect on their amplitude. Taken together, these results suggest that 5-HT suppresses glutamatergic neurotransmission in the BNST by activating presynaptic 5-HT(1B) receptors to decrease glutamate release from presynaptic terminals. This study illustrates a new pathway by which the activity of BNST neurons can be indirectly modulated by 5-HT, and suggests a potential new target for the development of novel treatments for depression and anxiety disorders.

Figure 1. 5-HT reduced the amplitude of eEPSCs in the BNST

Whole cell patch clamp recordings were made on BNST slices and EPSCs were evoked by stimulating the stria terminalis. Membrane potential was held at −60 mV. A) In one BNST_AL neuron, application of RS-CPP (10 μM) had no noticeable effect on the eEPSCs, whereas DNQX (20 μM) nearly completely blocked eEPSCs. Notice that the eEPSCs traces in the ACSF and RS-CPP were overlapped. B) In another BNST_AL neuron, RS-CPP application caused a leftward shift of the decay phase of eEPSCs, whereas did not affect the rising phase and peak amplitude of eEPSCs. Subsequently application of DNQX blocked the residual component of eEPSCs. Inset: the NMDA receptor-mediated EPSCs was revealed by subtracting the eEPSCs traces obtained before and during RS-CPP application. C) Representative traces showing the eEPSCs recordings before, during and after 5-HT application. 5-HT suppressed the amplitude of eEPSCs, an effect that reversed after 10 min of wash with ACSF. D) The time course of 5-HT (50 μM) action on eEPSCs amplitude indicated a fast activating and recovery pattern. **, p<0.01 vs baseline. E) Dose-response curve of the inhibiting effect of 5-HT on eEPSCs in BNST, the IC₅₀ was 4.0 μM. The numbers of neurons tested in each 5-HT concentration were between 6 and 10.

Figure 2. 5-HT_1B receptor agonist mimicked and 5-HT_1B antagonist blocked the effect of 5-HT on eEPSCs in BNST neurons

A) Representative traces showing application of sumatriptan (10 μM) or CP93129 (10 μM) reversibly reduced the amplitude of eEPSCs; the effect could be reversed after 10 min washout with ACSF. B) Group data showing 10 μM sumatriptan (n=11) and 10 μM CP93129 (n=8) significantly reduced the amplitude of eEPSCs, while 1 μM sumatriptan (n=6) only slightly reduced eEPSCs amplitude (p>0.05). C, D) Pre-application of 5-HT_1B antagonist GR55562 (10 μM) blocked the effect of 5-HT and sumatriptan on eEPSCs. **, p<0.01; ***, p<0.001

Figure 3. Suppression effect of 5-HT on eEPSCs could not be mimicked or blocked by 5-HT_1A receptor agonist or antagonist

A, B) 5-HT_1A receptor agonist, 8-OH DPAT (10 μM) has no apparent effect on eEPSCs. C, D) In the presence of 5-HT_1A receptor antagonist WAY100635 (200 nM), 5-HT (50 μM) suppressed the amplitude of eEPSCs (n=6, p<0.01), an effect showed no difference from that of 5-HT in control ACSF.

Figure 4. Sumatriptan has no effect on postsynaptic AMPA current induced by local pressure ejection of AMPA

Postsynaptic AMPA currents were induced by puff-application of AMPA (1 mM) onto recorded neurons every 1 min. A) Upper traces showed the inward currents induced by puff application of AMPA in ACSF and during sumatriptan application. B) No changes of postsynaptic AMPA current was observed during sumatriptan application.

Figure 5. 5-HT, sumatriptan and CP93129 increased the paired-pulse ratio

A) Increased PPR was observed associated with the decrease of eEPSCs amplitude. Group data showed the increased PPR during 5-HT (B), sumatriptan (C) and CP 93129 (D) application. Open circle showed the PPR of individual neurons, filled circle showed the mean PPR. *, ** paired t-test, p<0.05, 0.01 respectively.

Figure 6. Sumatriptan decreased the frequency of mEPCSs

Spontaneous mEPSCs in BNST were recorded in the presence of TTX (1 μM) and GABA_A antagonist SR 95531 (5 μM). A) Traces showing mEPSCs recorded from BNST neurons, and sumatriptan application reduced the frequency of mEPSCs (right). B) Cumulative plots showed the increase of inter-event interval of mEPSCs. C) Group data indicated sumatriptan reduced the frequency but have no effect on the amplitude of mEPSCs. n=11, **, paired t-test p<0.01

Figure 7. Schematic diagram of serotonergic modulation of neuronal excitability in the BNST

BNST receives glutamatergic inputs from key structures of limbic system and projects to downstream targets involved in stress response, addiction and reward system. Information integration in BNST can dramatically affect the function of downstream nuclei. Serotonergic inputs from caudal DRN innervate the BNST and modulate BNST neuronal excitability through pre- or postsynaptic mechanisms. Presynaptically, 5-HT activates 5-HT_1B receptor located on glutamatergic inputs to reduce the glutamate release, thus reduce the excitability of BNST neurons. Postsynaptically, activation of 5-HT_1A receptor inhibits and 5-HT_2A,_2C,₇ receptors excites BNST neurons.